FR2729392A1 - OLIGONUCLEOTIDES FOR THE DETECTION OF SALMONELLA - Google Patents

Abstract

A novel nucleotide sequence (I), involved in invasion of cultured HeLa cells by Salmonella enterica ssp. enterica serovar. typhi (S. typhi), is either the 97-1755 or 1766-2255 region (designated iagA and iagB, respectively) of an approx. 2.4 kb. sequence reproduced.

Description

OLIGONUCLEOTIDES FOR THE DETECTION OF SALMONELLA

  The genus Salmonella contains two species,

  Salmonella enterica, a species divided into six sub-

  species, on the basis of biochemical characters and

  homologies at the DNA level, and Salmonella bonqori.

  The genus is subdivided into more than 2000 serovarieties defined using somatic and flagellar antigens. Salmonella bacteria are generally pathogenic for animals or humans. Salmonella is known to be one of the most common food poisoning agents in developed countries; therefore, rapid and reliable detection methods for Salmonella subspecies are important. Salmonella responsible for toxi-infections

  mainly belong to the sub-

  species I (still called group I) of S. enterica.

  However, toxi-infections are not the only pathologies caused by

Salmonella.

  For example, Salmonella enterica subspecies enterica serovariety typhi (hereinafter referred to as Typhi)

  is the causative agent of human typhoid fever.

  Given the nature of infections caused by salmonella and the need in particular to find their presence in biological samples taken from patients or in food, it appears essential to have rapid and sensitive means to detect their presence. The standard culture methods widely used until now for the detection of salmonella require a long time and are not suitable for example for monitoring the contamination of food products. To overcome the disadvantages of these methods, several methods based on molecular biology techniques such as hybridization tests and polymerase chain reaction tests have already been proposed. Different DNA probes have been used in several hybridization and PCR protocols to detect subspecies of Salmonella in the diet. However, none of these techniques is completely satisfactory, since the sequences used are not fully known or not exclusively present in the genus Salmonella and thus, can lead to cross-reactions between the probe and DNA sequences of others. enterobacteria or can lead to a large number

  false negatives or false positives.

  The inventors have sought means allowing the specific and sensitive detection of all the

  Salmonella species S. enterica and / or S. bonqori.

  With this in mind, they were interested in the strain Salmonella enterica subspecies enterica serovariety typhi (S. Typhi) and the gene involved in the invasion of

cells by S. Typhi.

  In addition, they have defined certain conditions allowing the specific detection of specific groups of Salmonellae, for example Group I bacteria. It has already been shown in the prior art that the Typhi strain is capable of adhering to HeLa cell monolayers and enter these cells (Yabuuchi et al, 1986). However, until now, the genetic determinants involved in this process of adhesion and entry into cells have not been clearly identified. Elsinghorst et al (1989) cloned a Typhi chromosomal fragment, which gives Escherichia coli bacteria the

  ability to penetrate Henle 407 cells.

  reo-_S '... osc -......... JFl in the invasion of HeLa cells by Typhi strain

  Ty2 has been identified and cloned (Popoff and Dion, 1990).

  The inventors of the present application have identified on a 2.4 kb DNA fragment of S. typhi contained in the 7.9 kb HindIII sequence described by Popoff and Dion (1990), regions likely to participate in the invasion activity of Salmonella enterica subspecies enterica serovariété Typhi in cells, and in particular in cell cultures of HeLa type, these regions being more likely to be used in reactions for carrying out a generalized diagnosis of all representatives of S. enterica and / or S. bongori species or possibly under special detection conditions, for the

  specific diagnosis of group I of S. enterica.

  A sequence called IagA and a sequence called IagB have been identified by the inventors and characterized as participating in the cellular invasion manifesting during a Salmonella infection.

  enterica subspecies enterica serovariate Typhi.

  The specificity of these sequences within S. Typhi led the inventors to propose their use to define means for the diagnosis of S. typhi infection, or even for the diagnosis of an infection with Salmonella species S. enterica and / or S. bonqori or in some cases for

  highlighting S. enterica of specific groups.

  These means that can be used for the diagnosis of infection with Salmonella enterica and / or Salmonella bongori include oligonucleotides that can be used in amplification reactions of nucleotide sequences, for example polymerase chain reaction reactions. The invention also relates to probes for the detection of nucleic acids of S. enterica or a subspecies specificrmP cip. and / or S. bor..ri, these nucleic acids being the case of the amplified fragments. The invention also relates to a kit and a method for detecting the presence of Salmonella enterica and / or Salmonella bonqori in biological samples and, for example, in food products or in any sample subject to diagnosis. clinical. These detection methods and kits are, according to a particular embodiment of the invention, specific to the strains of the

group I of S. enterica.

  According to another embodiment of the invention, these methods allow on the contrary to search for the presence of S. enterica or S. bongori bacteria of the genus Salmonella. The Salmonella genus thus

  six subspecies or groups I, II, III, IV, V or VI.

  Subspecies I, II, III, IV and VI belong to the species S. enterica and the subspecies V belongs to

S. bonqori species.

  The invention also relates to the nucleotide sequences involved in the cell invasion by Salmonella enterica subspecies enterica serovariété Typhi, characterized in that it is one of the iagA or iagB sequences respectively between the nucleotides 97 and 1755 of the sequence shown in Figure 1 (IagA) and between nucleotides 1776 and

  2255 of the sequence shown in Figure 1 (IagB).

  The invention is also directed to nucleotide sequences modified with respect to iagA or iagB but nevertheless having the same properties as regards the invasion of the cells, or hybridizing under stringent conditions with one of the aforesaid sequences. The present application also relates to IagA and IagB proteins corresponding to the sequences shown in FIG. 1, or variants of these sequences which are obtained by amino acid to amino acid, provided that the sequence thus obtained is recognized by antibodies directed against one of the

said IagA or IagB sequences.

  In general, the subject of the invention is any amino acid sequence encoded by the iagA genes and

iagB shown in Figure 1.

  The invention also relates to any fragment of one of these sequences, sufficient to maintain S. typhi its adhesion and infection properties of cells, and in particular HeLa cells in culture. The method of infection of HeLa cells in culture is the standard method which has been described in particular in the published international patent application.

under number WO 92/01056.

  According to another aspect, the invention relates to means for the detection of the presence of S. enterica and / or S. bonqori and, where appropriate, for the quantification of S. enterica and / or S. bonqori in

biological samples.

  "Biological sample" means any sample taken for in vitro analysis in animals or humans or taken from food products of any kind or from any liquid, solid or gas which may contain the desired pathogens. The subject of the invention is, in this context, a nucleotide sequence comprising at least 9 nucleotides, characterized in that it hybridises with

  one of the IagA or IagB sequences presented above.

  The hybridization conditions mentioned above are defined according to the desired specificity of the hybridization and appropriate conditions are given as an indication.

  in the examples of this application.

  Preferably, the invention relates to oligonucleotides derived from the C-terminal part of the

  iagA sequence shown in Figure 1.

  Oligonucleotide-like sequences may be selected for use as primers for either amplified detection, genomic DNA or Salmonella cDNA of S. enterica and / or S. bonqori to the other groups I to VI or a part of these groups is under other conditions for the specific detection of S. enterica group I. It can be in particular nucleotide sequences obtained by chemical synthesis according to the known methods of the man

of career.

  Preferred oligonucleotides useful for the amplification of nucleic acid characteristic of bacteria belonging to one of the groups I, II, IIIa, IIIb, IV, V or VI of the genus Salmonella and in particular genomic DNA or the S. enterica and / or S. bonqori cDNAs are for example the following (their position within the IagA sequence shown in FIG. 1 being indicated): Iagl position: 5'-TA TTA AGT ATG CAG GTT ATG 3 ' 1424-1443 Iag2: 5'-AGA GAA TT CTG GAA AGT GAA-3 '1585-1605 Iag3: 5'-ATA TCC ACG CAG GAA ATA ACA GGA CTT-3' 1495-1521 Iag4: 5'-GAG CGT GCC TTA CGC ACG ATA-3 '1564-1584 Iag5: 5'-GCA GGG ATC ACT AAG CTG TG-3' 1318-1337 Iag6: 5'-CGT GGG CAA CCA GCA CTA ACG-3 '1637-1657 Slml: 5'- CG GGT TAA AGG TTA TCA CCT-3 '709-728 Slm2: 5'-AG CAT GGC GCA AAT GGG-3' 1014-1031 Slm3: 5'-GCA CCA GGA AAG CAT TAA GTT GAT AGA

ACA C-3 '732-762

  Slm4: 5'-CTT CGC TGG ACA GAC ACA CA-3 '823-842

SS28: 5'-TAA TGC TTT CCT GGT GC-3 '.

  Other oligonucleotides susceptible to be used as primers for the amplification of the DNA or cDNA of the iagB gene of all Salmonella strains of S. enterica and / or S. bonqori species were defined from of the iagB sequence shown in

Figure 1.

  The subject of the invention is therefore the oligonucleotides corresponding to the following sequences: Iag7: 5'-T ACG GCA TGG GCT GAT TGC T-3 'Iag8: 5'-T TAC GCT ATC GCC CAG CAG CAG GA-3' Iag9: 5 These oligonucleotides can also be used as probes, for example for the detection of cancer products, and the like. amplification of DNA and / or cDNA

of S. enterica and / or S. bonqori.

  A preferred pair of primers for carrying out the amplification of the nucleic acid of S. enterica and / or S. bonqori, whatever the group of membership of the bacterium, is for example formed

  primers Iag5 (sense) and Iag6 (antisense).

  This pair of primers directs the amplification of a

  nucleic acid fragment of 340 bp.

  Another preferred pair of primers is Slml (sense) and Slm2 (antisense) primers. These primers are likely to hybridize with the DNA or cDNA of S. enterica and / or S. bonqori bacteria of one of the

groups I, II, III, IV, V or VI.

  According to another preferred embodiment, the invention relates to oligonucleotides useful as primers for the specific detection of Salmonella enterica Group I when the detection conditions after the amplification of the DNA or cDNA are those described in Example I Such primers are characterized by their ability to amplify nucleic acid sequences of S. enterica or S. bonqori strains representative of groups I, II, III, IV, V and VI, but for which the detection conditions are those set forth in Example I, allowing the sole detection of group I bacteria. A pair of oligonucleotides that can be used for these purposes, as specific primers for the detection of DNA or DNA sequences. S. enterica cDNA group I is for example constituted by the following sequences: SS2 5'-CCGGGCAGATGATACCC-3 'and

SS28 '-TAATGCTTTCCTGGTGC-3'.

  The oligonucleotides defined by the inventors make it possible to envisage the diagnosis of S. enterica and / or S. bonqori under satisfactory conditions of sensitivity of speed, ease and specificity. The invention also relates to a kit for the detection of S. enterica and / or S. bonqori by amplification of genomic or complementary DNA of S. enterica and / or S. bonqori, characterized in that comprises: oligonucleotides as described above, capable of hybridizing under stringent conditions with the genomic DNA or the S. enterica and / or S. bonqori cDNA, a probe for the detection of the amplified fragments corresponding to the one of the definitions given in the previous pages, - the reagents necessary for carrying out the

amplification reaction.

  The invention therefore particularly relates to the use of the abovementioned oligonucleotides, as primers for the amplification of a DNA or cDNA sequence of Salmonella enterica and / or Salmonella bonqori, included in one of the iagA or iagB sequences as described in the preceding pages or comolementIirli AIiue tell Senc., or crcore the use of these oligonucleotides as probe for

  detection of an amplified nucleotide sequence.

  For example, the oligonucleotides iag5 and iag6 can be used respectively as sense and antisense primers for the detection of S. enterica and / or

  S. bonqori group I, II, III, IV, V or VI.

  Similarly, the pair of primers Slml and Slm2 can be used for the detection of bacteria of the species S. enterica and / or S. bonqori of one of these groups

in a biological sample.

  The invention also relates to the use of SS2 and SS28 oligonucleotides for the specific detection in vitro in a biological sample of S. enterica of group I. The detection is specific when the primers used for the amplification of the desired nucleotide sequences allow the amplification of S. enterica and / or S. bongori bacteria belonging to one of the other groups II, III, IV, V or VI, but that the conditions used do not allow the detection of the bacteria of these same groups or different organisms likely to be present

  in the biological sample tested.

  The invention thus relates to a set of oligonucleotides that can be used for the detection of S. enterica and / or S. bonqori bacteria, after amplification of the genomic or complementary DNA of S. enterica and / or S. bonqori, characterized in that it comprises: - a pair of oligonucleotides corresponding to the previously given definitions, capable of hybridizing under stringent conditions with the genomic DNA or the S. enterica and / or S. bongori cDNA, - a probe meeting the characteristics given above. A first set of usable oligonucleotides. i. t .... a biological ehantillo1, of strains of Salmonella enterica and / or S. bonqori belonging to one of groups I, II, III, IV, V or VI, is characterized in that it contains the oligonucleotides following: - the Iag5 sequence (5'-GCA GGG CTA ACT AAG CTG TG-3 'and the sequence Iag6 (5'-CGT GGG CAA CCA GCA CTA ACG-3') usable as primers for amplification and the Iag3 (5'-ATA TCC ACG CAG GAA ATA ACA GGA CTT -3 ') sequence which can be used as a revealing probe and the Iag4 sequence (5'-GAG CGT GCC TTA CCG ACG ATA-3')

usable as a capture probe.

  Another set of oligonucleotides that can be used for the specific in vitro detection in a biological sample of Group I S. enterica is characterized in that it comprises the following oligonucleotides: SS2 (5'-CCGGGCAGATGATACCC-3 'and

SS28 ('-ATATGCTTTCCTGGTGC-3').

  The present application also relates to an iagA protein encoded by the nucleotide sequence iagA shown in FIG. 1 and an iagB protein encoded by the iagB nucleotide sequence shown in FIG.

Figure 1.

  Preferably, the iagA and iagB proteins respectively have the amino acid sequences

shown in Figure 1.

  Also within the scope of the invention is a method for the in vitro detection in a biological sample of nucleotide sequences Salmonella enterica and / or S. bongori, previously amplified for example by PCR, characterized in that it comprises the steps of: denaturation of the S. enterica and / or amplified S. bonqori sequence, - contacting the nucleotide sequences

  amplified by S.Sc.

  he bongori, with a capture probe and a probe of

  revealed from the oligonucleotides

  defined above under conditions allowing the hybridization of said capture and revelation probes with the aforesaid amplified nucleotide sequence of S. enterica and / or S. bonqori, the capture probe being attached to the surface of a well of a microtiter plate and the revealing probe being labeled and free in a suitable hybridization buffer; incubating the reaction mixture for a time sufficient to allow the hybridization reaction; washing to remove unreacted oligonucleotides; - revelation of the revelation probes having

  hybrid to the amplified nucleotide sequences.

  The detection method described above can be advantageously characterized in that the detection is carried out according to the following steps: denaturation of a volume 41 of the amplified sequence by volume-to-volume addition of a solution mM NaOH, 40mM EDTA, prehybridization microplates whose well surface is coated with the capture probe, in a suitable hybridization buffer, - release of the microplate and filling each of the wells with 20041 hybridization buffer containing the denatured amplified fragment and the probe of peroxidase-labeled revelation at the concentration of 10 ng / l, incubation of the mixture for one hour at 37 ° C. with stirring, washing of the reaction mixture with a 10 × washing solution (10 μM Tris, 3 M NaCl, 1% Tween 20, pH 7.4), - detection of the activity of the peroxidase linked to the probe by colorimetry in the presence of a colored substrate. The revelation of the activity of the peroxidase present on the revelation probe can be obtained by carrying out the following steps: deposit of 20041 of a solution 40mM trisodium citrate, 0.03% H20 30%, 7.5mg / ml of orthophenylenediamine (OPD) in each of the wells containing the reaction mixture, incubation of the microplate for 30 min in the dark and at 37 ° C., blocking of the reaction by addition of 504 μl / well of a 4N H2SO4 solution - determination of the optical density at a

  wavelength of 492nm (reference at 620nm).

  Interestingly, the capture probe used is the oligonucleotide Iag4 and the probe of

  revelation is the oligonucleotide Iag3.

  Thus, the means defined in the context of the invention allow qualitative or quantitative detection of the presence of S. enterica and / or S. bongori bacteria, whether it is a non-specific detection in the breast. one of the groups I, II, III, IV, V or VI of S. enterica and / or S. bongori. Under specific conditions of implementation of the detection step, as described in Example I, primers SS2, SS28 and SS40 probe allow on the contrary the specific detection of

  Group I bacteria of S. enterica.

  Other characteristics and advantages of the invention appear in the examples which follow and in the figures: Figure i: Nucleotide sequence of a DNA fragment

  2.4 kb from the Salmonella invasion region ser.

  Typhi. Potential ribosome binding sites are underlined. Figure 2: Percentage of activity obtained with different strains of Salmonella belonging to

  different serovarieties by sandwich hybridization.

  Serovarieties of the different Salmonella isolates tested: a: S. enterica subspecies enterica (1), ref .: C53 b: S. enterica subspecies salamae (II), ref .: 975-71 c: S. enterica subspecies salamae (II), ref. : 3975-83 d: S. enterica subsp. Arizonae (IIIa), ref .: 1600 K e: S. enterica subsp. Arizonae (IIIa), ref .: So -20 f: S. enterica subspecies diarizonae (II) , ref .:

5250-85

  g: S. enterica subspecies diarizonae (IIIb), ref .:

8013-93

  h: S. enterica subspecies houtenae (IV), ref .: 1357-73 i: S. bongori, ref .: 2790-79 k: S. enterica subspecies indica (VI), ref .: 4355-84

  - 7, 6, 5, 4, and 3: log (amount of DNA molecules).

  Figure 3: Alignment of the sequences of the amplified fragments (nucleotides 1345 to 1644) of the 6 groups of Salmonellae. Figure 4: Amplification with primers Iag5 and Iag6 on two representatives of each group of Salmonella. Figure 5: Autoradiography of the Southern blot of

  amplified products of Salmonella.

  Figure 6: Determination of the minimum number of molecules

  of chromosomal DNA that can be detected.

  Autoradiography of Southern blot and microplate hybridization. Figure 7: Location of Oligonucleotides

  selected within the IagA gene.

EXAMPLE I

  CLONING AND SEQUENCING THE 2.4 kb DNA FRAGMENT This DNA fragment was subcloned using a restriction fragment obtained by cleavage with the HindIII enzymes, from the 7.9 kb HindIII sequence described in the publication of Popoff and Dion, 1990, in derivatives of the vector m13 (Messing and Vieira,

1982).

  After performing this cloning, the chain termination dideoxy method was carried out using the modified T7 DNA polymerase (Sequenase, USB Corp.) and universal synthetic oligonucleotides as primers. All the ends of the restriction fragments used overlapped each other. DNA sequencing was performed at least 2 times on each strand. The nucleotide sequence was analyzed using the Lipan program and

Pearson, 1985.

  As shown in the sequence shown in FIG. 1, two open reading phases are contained in the sequence fragment; they are designated by the terms iagA (abbreviation of invasion associate gene) and iagB. The two open reading phases are transcribed in the same orientation. The first ATG codon (bp 97) of the open reading phase of iagA, which is preceded by the 5'-AGAGA-3 'sequence, is assumed to correspond to the translation initiation site of the iagA gene. The iagA gene encodes a polypeptide comprising 553 amino acid residues with a calculated molecular weight of 63026 Da. Homology

  significant was detected between the N-domain

  of the IagA protein and the domain corresponding to the PhoB transcriptional regulatory protein (24% identity and 52% similarity for a superposition of 108 amino acids) and the PhoP protein (25% identity and 69% of similarity for aligned amino acids) of E. coli. The ATG initiation codon of the iagB gene (bp 1776) is also preceded by a potential ribosome binding site (5'-AGGAAG-3 '). The iagB gene encodes a polypeptide having 160 amino acids and having a calculated molecular weight of 18369 Da. The comparison of the sequence of the IagB protein with the translated sequences contained in the Genbank database showed a significant homology with the IpgF protein (43% identity and 66% similarity for 151

amino acids aligned).

  The IpgF protein is encoded by the ipgF gene which is located on the plasmid associated with the virulence of Shiqella flexneri, at the 5 'end of the mxi-spa locus.

(Allaoui et al, 1993).

  The proteins found in Salmonella enterica subspecies enterica serovariété Typhi would therefore have a role in the infection by these bacteria, and in particular in the adhesion and

penetration into the cells.

EXAMPLE 2

  SPECIFIC DETECTION OF S. ENTERICA GROUP I

  A protocol for detecting subspecies of Salmonella by polymerase chain reaction (PCR) has been developed. A pair of oligonucleotides used as primer was defined to amplify a 93 bp fragment of a gene required for HeLa cell invasion by S. typhi strain Ty2. The amplification product was analyzed by non-radioactive sandwich hybridization on microtiter plates using two different oligonucleotides according to the procedure described by Chevrier et al.

  al, 1993, Mol. Cell. Probes 7, 187-197.

  The capture oligonucleotide was phosphorylated at its 5 'end and covalently bound to wells carrying amino groups of a microtiter plate. The detection oligonucleotide has been

  amine at its 5 'end and then labeled with a biotinyl-

  N-hydroxy-succinimide ester. After hybridization, the hybrid molecules were detected by avidin conjugated with alkaline phosphatase and a chromogenic substrate. This method only requires the use of a thermal cycler and a conventional microtiter reader, and can be implemented

on a large scale.

MATERIALS AND METHODS

  Bacterial strains Two hundred and twenty-eight clinical isolates (Table 1) including S. bongori (Sambrook et al., 1989, Molecular cloning, Cold spring Harbor Laboratory Press, Cold Spring Harbor, NY), S. enterica subspecies I ( 116), II (56), IIIa (11), IIIb (30), IV (5) and VI (5) and 16 strains of non-salmonella Enterobacteria (Table 2) representing 9 different genera were used in this study . The strain C53 of S. ser. Typhimurium was used as a positive control, and E. coli strain HB101 was used as a negative control in

PCR tests.

  DNA extraction The strains were cultured on LB medium at 37 ° C. In order to carry out the rapid extraction of the DNA, 2 ml of the culture kept overnight were centrifuged and resuspended in 1 ml of TE (buffer mM Tris-HCl pH 8 containing 1 mM EDTA). The cells were centrifuged, the pellet was resuspended in 500 g of sterile distilled water and heated at 100 ° C. for 10 minutes. Finally, the solution was centrifuged and the supernatant was

  preserved for PCR experiments.

  Oligonucleotide primers and probes Oligonucleotides were summed in a cyclone DNA synthesizer (Millipore-Waters)

  using phosphoramidite technology.

  The sequences of the oligonucleotide primers were as follows: SS2: 5'-CCGGGCAGATGATACCC-3 'and

SS28: 5'-TAATGCTTTCCTGGTGC-3 '.

  The oligonucleotide capture probe,

  SS40: 5'-CCCGAACTATCTCGATCTGTACAATATTATCATT-3 '

  was phosphorylated at its 5 'end with T4

  polynucleotide kinase (Boehringer) according to the description

  made by Sambrook et al., 1989, (Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The octadecanucleotide detection probe SS41 (5'-GCAGGTGATAACCTTTAA-3 ') was synthesized with an amino function at its end using the solid phase phosphoramidite method in an Applied Biosystem 380B DNA synthesizer and then labeled with D-DNA. biotinyl-2-aminocaproic acid-N-hydroxysuccinimide ester (Boehringer) according to

  description made by Tham et al, 1990, (FEMS

  Microbiol. Lett. 69, 109-116). The capture and detection oligonucleotides were both purified on a HR 10/10 rapid desalination column with

FPLC system (Pharmacia).

  PCR Experiments The amplification reactions were carried out in a total volume of 100 μl in a 50 mM Tris-HCl buffer pH 8.5, containing 4 mM MgCl2, 100 μg / ml bovine serum alumina, 1 μM of each primer, 200 μM of each dNTP and 1 U of Taq DNA polymerase (Amersham). The amplification mixture was overlaid with 100 l of mineral oil and subjected to 10 cycles of amplification

  according to the following description: the samples have

  were incubated at 94 ° C. for 10 sec to denature the DNA, at 60 ° C. for 10 seconds to match the primers to the DNA and at 72 ° C. for 30 sec. to carry out the extension reaction of the matched primers, this being followed by 30 cycles according to the following protocol: denaturation at 87 ° C. for 10 sec., pairing at 60 ° C.

  for 10 sec. and extension at 72 C for 30 sec.

  Thermal cycles were carried out in a set

  programmable heater (Thermal ractor Hybaid, UK).

  PCR experiments were performed with 5g1 of DNA solution. Each experiment included negative controls (5 41 of TE buffer) for each

  group of 10 samples and at the end of each series.

  Sandwich hybridization tests on CovaLink NH bands Two non-radioactive hybridization protocols on CovaLink NH bands have already been described by

  Chevrier et al (1993, Mol Cell Probes 7, 187-197).

  In the present case, a sandwich hybridization technique has been used insofar as it

  allowed a better sensitivity of the detection.

  The reaction was performed on microwells coated with SS40 oligonucleotide using a covalent binding procedure as described in detail by Chevrier et al. Or Rasmussen, S.R. et al.

  (1991, Anal Biochem 198, 138-142).

  The DNA fragment subjected to the PCR reaction was denatured directly into the wells by sequentially adding 95 1 of distilled water, 541 of PCR sample, 40 41 of detection probe and 14 41 of 1 N NaOH. per well. After 10 minutes, the neutralization was carried out by adding 21 g of 1 M NaH 2 PO 4 containing 1% of sarkosyl. All samples were made in duplicate. After neutralization, the strip was deposited on a metal surface and kept in an oven overnight at C. The final concentration of the biotinylated detection probe SS41 was 0.5 nM. During incubation in the oven, it is best not to leave unused wells empty, but to fill them with water

  in order to obtain homogeneous heat exchanges.

  The microwells were washed 5 times at room temperature with TBS-Tw (0.15 M NaCl, 10 mM Tris HCl buffer pH 8, 1% Tween 20). 100 μl of alkaline phosphatase-extravidin conjugate (Sigma) diluted to 14 g / ml in TBS-Tw containing 1% bovine serum albumin was added per well. Then, the band was incubated at room temperature for 1 h, washed 5 times with TBS-Tw, and finally 200 μl 1 M diethanolamine at pH 9.8 containing 1 mM MgCl 2 and 1 mM para-nitrophenyl phosphate were added. added. The reaction of the enzyme has

  been driven for 30 minutes to 2 hours.

  Absorbance was measured at 405 nm using a microplate reader (Dynatech). The signal obtained with the standard solution of the amplified DNA fragment (800 fm / well) of S. ser. Typhimurium strain C53 was considered 100% representative and used as a reference for each hybridization assay. Blank values correspond to the average absorbance measured in wells coated with oligonucleotide SS40 incubated only with 0.5 nM probe

  oligonucleotide SS41 biotinylated.

RESULTS

  Optimization of the method The primers and probes were chosen in the iagA sequence. Different pairs of primers were tested to optimize the sandwich hybridization technique on CovaLink microplates. The chosen pair of primers (SS2 and SS28) allowed the specific amplification of the 93 bp region of genomic Salmonella DNA. Using this pair of primers, it was found that a standard concentration of MgCl2 (1.5-2 mM) led to a relatively uninteresting amplification result and that a concentration of 4 mM in MgCl 2 was necessary. to obtain efficient amplification. Internal oligonucleotides, SS40 and SS41, were used in a non-radioactive hybridization assay as a probe of

  capture and detection probe respectively.

  Specificity of the technique The specificity of the method for the detection of salmonella was evaluated with 228 strains of Salmonella (Table 1) and 16 strains of heterologous bacteria (Table 2). The results are summarized in Table 3. Edwardsiella tarda, Klebsiella pneumoniae, Enterobacter and Acinetobacter species, Pasteurella, Vibrio harveyi, Serratia marcescens and more importantly Citrobacter species and all E. coli gave a hybridization signal less than 20%. Based on this value, it was concluded that all strains of Salmonella belonging to subspecies I could be detected by this method. In addition, only one strain (strain 3975-83) of the 56 strains of subspecies II and 3 strains of the 11 strains of

  Subspecies IIIa gave a positive signal.

  Salmonella bonqori and strains belonging to

  subspecies IIIb, IV and VI were not detectable.

  Level of Detection of the Technique with Whole Bacteria 1 / 10th dilution of a suspension of strain C53 of S. ser. Typhimurium (from 109 to 10-2 cells / ml) were made to estimate the minimum number of bacteria that could be detected by PCR followed by non-radioactive hybridization technique. DNA was extracted from each calibrated suspension, using the rapid extraction technique by boiling. The results obtained clearly show that the technique of rapid extraction of DNA by simply boiling the suspension before the PCR reaction is an efficient technique. Indeed, it allows the detection of only one cfu unit.

Table 1:

  Salmonella subspecies used to evaluate the specificity of DNA hybridization assays.

  Microorganism tested N0 of serovar N isolates Salmonella enterica subsp. Enterica I 116 43 serovar Adelaïde 1 Agona 2 Altona 1 Angoda 1 Bardo 2 Blocklev 1 Boxismorbificans 3 Braenderup 4 Brandenburg 1 Bredenev 1 Broughton 2 Cerro 1 Chester 1 Coeln 1 Concord 1 Dakar 1 Derbv 2 Enteridis 28 Georgia 1 Hadar 1 Heidelberg 4 Ibadan 2 Indiana 1 Infantis 5 Lexington 1 London I Mbandaka 1 Montevideo 6 Moscow 1 Ohio 1 Orion 1 Panama 3 Paratyphi B 2 Saintpaul 1 Typhimuriumn 13 Tvphisuis 1 Vaertan 1 Veneziana 1 Vinohrad) 1 Virchow 10 Wien ! WoodinNille 1 Yolo 1 Salmonella enterica subsp salamae II 56 56 Salmonella enterica subsp arizonae IIIa II Salmhnonella enterica subsp diarlzonae IIIb Salmonella enterica subsp houtenae IV Salmonella enterica subsp indica VI 5 Sa / nonella bongori 5 (initially S. enterica subspecies bongori V)

Table 2:

  Heterologous bacteria used in the DNA hybridization test Genus Species Number of isolates Escherichia coli 4 Edwarsie / la tarda 1 Citrobacter amalonaticus 1 freundii 1 Klebsiella pneumoniae 1 Enterobacter agglomerans 1 asburiae 1 hormoechei 1 Pasteurella multocida 1 Acinetobacter Iwoffii 1 haemolyticus 1 Vibrio harveyi 1 Serratia marcescens 1

Table 3:

  Clinical strains of bacteria and controls tested in a hybridization assay Sandwich S.enterica S. enterica S. enterica S. enterica S. enterica S. enterica S. bongori No Subsp. subsp. subsp. subsp subsp. subsp. Salmonella without DNA enterica salamae arizonae diarizonae houtenae indica activity (%)

% -20% 116 1 3 0 0 0 0 0 0

19% 0 51 8 12 4 4 5 9 0

  > white <white 0 4 0 18 1 1 0 7 23 Total 116 56 1 1 30 5 5 5 16 23 Quantitative hybridization with purified genomic DNA The non-radioactive hybridization procedure used in the tests reported here can easily be implemented in quantitative studies. To compare the hybridization signals obtained with different strains of Salmonella, the DNA

  extracted from 10 strains representing the 6 subtypes

  Salmonella enterica species and Salmonella bonqori species, then calibrated amounts of DNA were subjected to PCR reactions followed by sandwich hybridization. The results are reported in FIG. 2. It has been demonstrated that the hybridization signal obtained with 107 Salmonella bonqori DNA molecules or subspecies II, IIIa, IIIb, IV and VI of Salmonella enterica is weaker than the hybridization signal observed with 103 DNA molecules of the strains of subspecies I. However, it is important to note that isolate 3975-83 (subspecies II) gave the same hybridization signal as strains belonging to subspecies I.

DISCUSSION

  PCR amplification allows very sensitive detection of specific DNA sequences. The sensitivity of the amplification depends essentially on the number of copies of the target DNA, the purity of the sample to be analyzed, the DNA extraction method, and the sensitivity of the method used to detect PCR products. Visualization of PCR products by ethidium bromide staining in an electrophoresis gel is not compatible with the routine use of the art and is not sufficiently sensitive. Sensitivity can be enhanced by the use of double PCR or DNA probes with Dot blot or Southern blot hybridization. It is sensitive to DNA contamination and Dot blot or Southern blot hybridization techniques are not available.

  are not appropriate for automation.

  Microplate hybridization thus provides an appropriate technique for the detection and quantification of amplified fragments by PCR. The simple covalent attachment of nucleic acids to microwells represents an interesting alternative to passive adsorption and a significant improvement for the detection of amplified PCR fragments on microwells. It is known that strains of Salmonella causing infections in humans are essentially subspecies I. In fact, more than% of the clinical isolates in humans belong to this subspecies (Rowe, B., 1987 , Salmonella surveillance, World Health Organization London). Moreover, in 1991, the "National Center for Veterinary and Food Studies" in Paris (France) reported [Corbion, B. et al., 1991, Inventory of Salmonella] that in previous years, most of the strains isolated at animal in food or in the environment in 1988 and 1989 (ie, 18832 strains) belongs to

subspecies I (99.2%).

  The results reported here made it possible to define a method based on PCR amplification for the detection of pathogenic strains of Salmonella. A pair of primers, SS2 and SS28, and a pair of probes, SS40 and SS41, were selected from a gene necessary for Salmonella ser invasion of HeLa cells. Typhi strain Ty2. Using the combination of the PCR technique and non-radioactive sandwich hybridization on a microplate, all

  Salmonella subspecies I were detected.

  The limit of detection was below a threshold of 10 cells per PCR tube, which is consistent with results obtained by other similar PCR techniques. Given the kinship of nucleic acids between members of enterobacteria, it was important to control the specificity of these new primers and probes with the kinds of enterobacteria most likely to lead to "false-positive" reactions. Results

  obtained, it can be concluded that no false reaction

  positive can not take place when the conditions of PCR and hybridization described above are followed. It is interesting to note that Salmonella strain 3975-83 (subspecies II) showed a hybridization signal identical to that obtained with isolates belonging to subspecies I. This strain was isolated in 1983 from stool of a human patient in Britain. On the basis of the biochemical characteristics, this new serovariety was classified in subspecies II but was considered an atypical strain since its presence in gelatinase was not detected (Le Minor, L. et al, 1984, Supplement No. XXVII, 1983, to Kauffmann-White Scheme, Ann Microbiol (Institut Pasteur) 135 B, 45-51). In light of the results reported here, the taxonomic position of strain 3975-83 should be re-examined using the

DNA-DNA hybridization technique.

  The data presented here indicate that the hybridization method based on the use of a gene necessary for the invasion of HeLa cells by Salmonella ser. Typhi strain Ty2 can distinguish strains of subspecies I from Salmonella from other enteric bacteria, including E. coli. Non-radioactive hybridization on a Covalink NH microplate is sensitive and suitable for the analysis of a large number of samples.

  EXAMPLE 3 DETECTION OF SALMONELLA AMPLIFIED DNA

BY SANDWICH HYBRIDIZATION

  Sequence of Oligonucleotides The selected DNA fragments are as follows (see position in the sequence of Figure 1): Terminal Part Iagl: 5'-TA TTA AGT ATG CAG GTT ATG-3 '1424-1443 Iag2: 5'- AGA GAA TTT CTG GAA AGT GAA-3 '1585-1605 Iag3: 5'-ATA TCC ACG CAG GAA ATA ACA GGA CTT -3' 1495-1521 Iag4: 5'-GAG CGT GCC TTA CCG ACG ATA-3 '1564- 1584 Iag5: 5'-GCA GGG ATC ACT AAG CTG TG-3 '1318-1337 Iag6: 5'-CGT GGG CAA CCA GCA CTA ACG-3' 1637-1657 Slml: 5'-CG GGT TAA AGG TTA TCA CCT- 3 '709-728 Slm2: 5'-AG CAT GGC GCA AAT GGG-3' 1014-1031 Slm3: 5'-GCA CCA GGA AAG CAT TAA GTT GAT AGA

ACA C-3 '732-762

  Slm4: 5'-CTT CGC TGG GAC ACA AAG CA-3 '823-842 Preferentially, the pair of primers Iag5 (sense) and Iag6 (antisense) directs the amplification of a fragment of 340bp, the Slml pair (sense ) and Slm2 (antisense) directs the amplification of a fragment of

323bp (Figure 3).

  FIG. 4 shows the efficiency of the amplification of the pair of primers Iag5 and Iag6 on 2 representatives of

each group of Salmonella.

Detection method

  A sandwich hybridization detection format has

been used.

  Two oligonucleotides hybridize simultaneously to the denatured amplified fragment. One of them called capture probe is passively (but may also be covalently attached) attached to the surface of a 96 well microtiter plate well. The other called the revealing probe is marked by an element easy to highlight. The revealing probe is free in the

hybridization buffer.

  The capture and revelation probes are complementary to 2 different regions located in

inside the amplified fragment.

  The detection probe in the case described here, is linked to an enzymatic marker including a peroxidase and will serve as a revelation probe. It's the case

  preferentially oligonucleotides Iag3 and Slm3.

  Other oligonucleotides can be fixed to a solid support of the microplate type, a particulate or membrane support and serve as a capture probe, this particularly for the

oligonucleotides Iag4 and Slm4.

  Experimental Conditions: 1) Preparation of Salmonella DNA By the boiling method in the presence of Chelex (Chelex 6%, SDS 0.1%, NP40 1%, Tween 20 1%), the DNA sequences are obtained. This reagent is marketed by

  Biorad and used according to the manufacturer's protocol (ref.

  Walsh et al. 1991. BioTechniques 10: 506-513).

  2) Amplification According to the method initially described by Saiki and as described for example in the European patent EP

0201184.

  The PCR is carried out using the following reaction mixture: mM KCl mM Tris-HCl pH 8.3 1.5mM MgCl2 4M deoxyribonucleotides (dCTP, dATP, dGTP) 250gM UTP pmol of each of the primers 10ng DNA 1 unit of Uracyl N glycosylase

1 unit of Taq polymerase.

  The reaction mixture was made using 1041 of the solution containing the DNA to be amplified in a volume of 10041. The dUTP and the UNG are used in decontamination system (Patent Life Technologies European Patent Application 0 401 037). The thermocycler

  used is the 9600 Perkin Elmer.

  After incubation at 50 ° C. for 2 minutes to allow the action of UNG and denaturation at 95 ° C. for 5 minutes, the temperature cycles used are as follows: 5 cycles (95 ° C., 50 ° C., 15 sec. 72 C 15 sec) - 35 cycles (95 C 15 sec, 57 C 15 sec, 72 C 15 sec) 3) Visualization of the amplification reaction 3-1) Labeling of the revealing probe The probes are labeled with peroxidase of horseradish (ref PCR protocols: a guide to methods and application, Academic press (1990), 15, p4513-4534) and

  the activity of the enzyme is revealed in colorimetry.

  3-2) BET Colored Agarose Gel and Membrane Hybridization After amplification, 1041 of the amplification product are deposited on agarose gel and the DNA is transferred to membrane according to standard techniques (Maniatis). The membrane is prehybridized, 30 min at 68 ° C. in hybridization buffer (10X Denhart, 6 × SSC, 0.1% SDS) and then hybridized at 42 ° C. for 3 hours with 60 ° C.

  ng of probe per ml of hybridization buffer.

  Washing is then carried out according to the following steps: 2 × 10 min in 2 × SSC -0.1% SDS at room temperature, × 1 × 30 min in 0.1 × SSC 0.1% SDS at 42 ° C.,

  - 2 times 10 min in 2 X SSC at room temperature.

  Revelation: The membrane is sponged between two sheets of absorbent paper (Whatman 3MM paper) and

* placed in a clean and dry bin.

  The detection reagent Amersham (detection reagent ECL RPN 2105) is prepared extemporaneously volume to volume; 30 ml of total volume for a 5 x 8 cm membrane. An autoradiography cassette is obtained by attaching a sheet of absorbent paper (Whatman 3MM paper) to the bottom. All these steps can

  to be in the light. Then in darkroom.

  The membrane is bathed in the detection reagent for 1 min, DNA side up, the membrane is rapidly drained, it is placed in the cassette, DNA side up, a transparent plastic sheet is placed on top of it (otherwise the membrane sticks on the film) and we put a radiographic film over

(X-OMAT KODAK movie).

  The exposure is carried out for 30 min at room temperature then the film is developed by conventional revelation techniques (developer, water, fixer). 3-3) Microplate 3-3-1) Coating of the capture oligonucleotide It can be done by adsorption (Cook et al., NAR, 16: 4077-4095 (1988) or covalent coupling (Rasmussen, SR et al., 1991). Analytical Biochemistry 198,

138-142).

  3-3-2) Microplate hybridization and reading of the amplification product were denatured by volume-to-volume addition of a 200mM NaOH solution,

4OmM EDTA.

  The microplates, the well surface of which is coated with the capture probe, were prehybridized in hybridization buffer containing 10XDenhart,

6XSSC, 0.1% SDS.

  The microplate was then emptied and each of the wells received 20041 of hybridization buffer containing the denatured amplified fragment and the 10 ng / 41l probe. Incubation took place

  for one hour at 37 ° C and stirring.

  After washing (10X wash solution: 100mM Tris, 3M NaCl, 1% Tween 20, pH 7.4), probe-bound peroxidase activity was detected in

  colorimetry in the presence of a colored substrate.

  To do this, 20041 of a 40mM trisodium citrate solution, 0.03% H20 30%, 7.5 mg / ml of orthophenylenediamine (OPD) were distributed in each of the wells. The microplate was incubated for 30 min in the dark at 37 C. 5041 / well of a 4N solution

  H2SO4 were added to block the reaction.

  The optical density was determined at a

  wavelength of 492nm (reference at 620nm).

  4) Sequencing of PCR products and alignment

manual of sequences.

  According to conventional techniques, using for example an automaton "373 DNA sequencer" from Applied

  Biosystem and the Applied dye terminator kit.

  Results The exemplified model is preferably the following oligonucleotide system: Iag5 primer sense-Iag6 antisense primer

  Iag3 revelation probe and Iag4 capture probe.

  (It should be noted that Iag4 can just as easily be

  labeled and used as a revealing probe).

  Specificity study It covered all strains

  listed in Tables 4 and 5.

  The amplification of the DNA extracted from the 45 strains of Salmonella tested generated a fragment of the expected size (see Fig. 5). Southern blots of all the amplified products were hybridized with the peroxidase-labeled internal Iag 3 oligonucleotide probe. None of the non-salmonella strains gave rise to hybridization with a peroxidase probe carried out on a membrane according to the protocol described above. The same amplification products were tested

in microplate format.

  The cut-off was arbitrarily fixed at 0.050. All representatives of each group of Salmonella give a higher optical density value than

0.050 (Table 6).

  Sensitivity To determine the minimum number of Salmonella chromosomal DNA molecules that can be detected, a purified chromosomal DNA dilution range has been amplified. 5 molecules are visible on Southern blot autoradiography and detected in microplate hybridization: the value obtained in

  Colorimetry is superior to Cut-Off (Figure 6).

  The oligonucleotides selected for carrying out this example were located on the

IagA gene sequence (FIG. 7).

STUDY SALMONELLA STRAINS

  No [Strains Serotype Group 1 Sa / monella Marseil // 2 Salmonella Nyanza I 3 Salmonella Poona 4 Salmonella Kampala Salmonella Taksony I 6 Salmonella Teshie I 7 Salmonella Indiana I 8 Salmonella enteritidis I 9 Salmonella Kentucky I Salmonella Napoli 11 841 1 1 a : d: in 215 I

12 1703 K 41: 2: 15 I

13 950 - 71 43: d: z39 I

14 10-65 44: 24, 223: - I

  3209-81 45: z 23 I 16 5331/86 62: z 29: - lia 17 3064-4 / 252 41: k: - Illa 18 594-54 38: z 54: - lila 19 1694 cdai 426 63: z 4 , z32: - Ilila 50/16 62: f, z 51: - lia 21 5251-85 58: r: z 53 Illb 22 1758-76 6,14: z 10: enx 215 I IIb 23 453-68 16: liv: z53 Ilib 24 4305-57 1 6: li (v): z 35 IlIlb 1698-75 1: liv: z Illb 26 8275-94 47: r: enx 215 IlIlb 27 8283- 94 53: z 10: z lilb 28 cdc 456-5 / 93 40: i: 1, 5, 7 IIIb 29 8284-94 60: i: z lilb 1 693 K 38: k: z 55 Ilib 31 170748 f: z 51: - IV 32 7231/89 45: z 36, z 38 IV 33 6887/6048: f, z51: - IV 34 1357/73 43: z4, z 24: - IV 1550K 16: z4, z23: - IV 36 Salmonella Bongor 261 -66 48: z35: - V 37 Salmonella Camdeni 2022 - 77 44: r: - V 38 4985 - 85 48: z39: - V 39 7688 - 9166: z39: V 1387-7340: a: - V 41 1941 - 77 6.7: z 41: 1.7 Vl 42 1449 K45 a en Vl 43 4355 84 1,6,14,25 a: en, x VI 44 1711 K 11b enx VlI 1688 K 1,6,14,25 Z 10 1, 12.7 Vl

TABLE 4

NON-SALMONAL STRAINS

  No. Name I Identification 1 Klebsiel / a oxytoca 0059 SDP 2 Klebsiella pneumoniae 0054 SDP 3 Acinetobacter baumanii 0033 SDP 4 Proteus mirabilis RP402 Serratia marcescens 0042 SDP 6 Enterobacter agglomerans 0067 SDP 7 Citrobacter diversus 0068 SDP 8 Pseudomonas aerugminosa 0011 SDP 9 Enterobacter aerogenes 0066 SDP Escherichia coli0131 SDP 11 Enterococcus faeca / is 76117 12 Proteus mirabilis AP03 13 Enterococcus faecalis 76117 14 Enterobacter cloacae 0060 SDP Mycobacterium avium 6 16 Mycobacterium tuberculosis H 37 RV 17 Listeria monocytogenes 1/2 LG3

TABLE 5

DETECTION ON MICROPLATE

  SAMPLE DO at 420 nm 2 Nyanza gpe I 3.029 3 Poona gpe I 3.103 11 gp Il 3.155 12 gp Il 0.751 18 gp iII a 3. 139 gp III a 3.068 21 gp III b 3.161 gp III b 3. 201 31 gp IV 0.272 gp IV 0.527 36 gp V 1.868 gp V 3.347 gp VI 0.900 Klebsie / oxytoca 0.022 Klebsie / pneumoniae O. 017 Acinetobacter baumanii 0.024 Proteus mirabilis 0.019 Serratia marcescens 0.019 Enterobacter agglomerans 0.023 Mycobacterium avium n 6 0.025 Mvcobacterium tuberculosis H 37 RV 0.020 Listeria monocytogenes 1 / 2 LG3 0.015 Water indicator 0.018 Water indicator 0. 022

TABLE 6

Claims (19)

  1. Nucleotide sequence involved in the invasion of cultured HeLa cells by bacteria of the species Salmonella enterica subspecies enterica serovariété Typhi (called S. Typhi), characterized in that it is the iagA sequence between nucleotides 97 and 1755 of the sequence shown in Figure 1 or iagB between nucleotides 1776 and 2255 of the sequence shown in Figure 1.   2. Oligonucleotide derived from a sequence according to claim 1, characterized in that it corresponds to one of the following sequences: Iagl: 5'TA TTA AGT ATG CAG GTT ATG-3 'Iag2: 5'-AGA GAA TT CTG GAA AGT GAA-3 'Iag3: 5'-ATA TCC ACG CAG GAA ATA ACA GGA CTT -3' Iag4: 5'-GAG CGT GCC TTA CCG ACG ATA-3 'Iag5: 5'-GCA GGG ATC ACT AAG CTG TG-3 'Iag6: 5'-CGT GGG CAA CCA GCA CTA ACG-3' Slml: 5'-CG GGT TAA AGG TTA TCA CCT-3 '1Slm2: 5'-AG CAT GGC GCA AAT GGG-3' Slm3: 5'-GCA CCA GGA AAG CAT TAA GTT GAT AGA ACA C-3 '1Slm4: 5'-CTT CGC TGG GAC ACA AAG CA-3' SS28: 5'-TAA TGC TTT CCT GGT GC-3 '.   Iag7: 5'-T ACG GCA TGG GCT GAT TGC T-3 'Iag8: 5'-T TAC GCT ATC GCC CAG CAG CAG GA-3' Iag9: 5'-T GGT CAT AAC CGA GAT GGT TCA ACC GAT C- 3 'IAG10: 5'-A CAG TTG TTA CAG GAT CCC T-3' 3. An oligonucleotide characterized in that it is derived from the nucleotide sequence IagA according to claim 1 and in that it is selected from the sequences that can be used as primers for the specific detection of Salmonella enterica of group I or for the detection of a nucleotide sequence thus amplified.   4. Oligonucleotide according to claim 3, characterized in that it corresponds to the following sequence: SS28: 5'-TAA TGC TTT CCT GGT GC-3 '.   5. Pair of oligonucleotides according to claim 2, characterized in that it is one of the following pairs: Iag5 (direction): 5'-GCA GGG ATC ACT AAG CTG TG-3 'and Iag6 (antisense) ): 5'-CGT GGG CAA CCA GCA CTA ACG-3 ', or Slml (sense): 5'-CG GGT TAA AGG TTA TCA CCT-3'and   Slm2 (antisense): 5'-AG GGC CAT GCA AAT GGG-3 '.   6. Sequence of nucleotides capable of hybridizing with Salmonella enterica and / or Salmonella bonqori, characterized in that it corresponds to one of the following sequences and in that it is labeled: Iag3: 5'-ATA TCC ACG CAG GAA ATA ACA GGA CTT -3 'or Iag4: 5'-GAG CGT GCC TTA CCG ACG ATA-3' or Slm3: 5'-GCA CCA GGA AAG CAT TAA GTT GAT AGA ACA C-3 'or   Slm4: 5'-CTT CGC TGG ACA GAC ACA CA-3 '.   7. Sequence of nucleotides according to claim 6 (or probe), characterized in that the probe Iag3 or the probe Slm3 is a probe for revealing the presence of an amplified nucleotide sequence and that the probe Iag4 or the probe Slm4 is a probe of   capturing an amplified nucleotide sequence.   8. Use of the oligonucleotides according to claim 2, as primers for the amplification of a DNA or cDNA sequence of Salmonella enterica and / or Salmonella bonqori included in a nucleotide sequence according to claim 1 or complementary to such a sequence, or as a probe for the detection of an amplified nucleotide sequence. 9. Use of the pairs of oligonucleotides according to claim 5, as primers for the amplification of nucleotide sequences of a bacterium of the species S. enterica and / or S. bonqori, group I, II, III, IV, V or VI.   10. Set of oligonucleotides usable for the detection of S. enterica and / or S. bonqori bacteria after amplification of the genomic or complementary DNA of S. enterica and / or S. bongori, characterized in that it comprises: a pair of oligonucleotides according to one   any of claims 4 or 5, capable of   to hybridize under stringent conditions with genomic DNA or S. enterica and / or S. bongori cDNA,   a probe according to claim 6.   11. A set of oligonucleotides useful for the in vitro detection in a biological sample of strains of Salmonella enterica and / or Salmonella bonqori belonging to one of groups I, II, III, IV, V or VI, characterized in that that it contains the following oligonucleotides: the Iag5 sequence (5'-GCA GGG ATC ACT AAG CTG TG-3 'and Iag6 (5'-CGT GGG CAA CCA GCA CTA ACG-3') usable as primers for the amplification and the sequence Iag3 (5'-ATA TCC ACG CAG GAA ATA ACA CGA CTT -3 ') usable as a revelation probe and the sequence Iag4 (5'-GAG CGT GCC TTA CCG ACG ATA-3') usable as a capture probe.   12. Set of oligonucleotides usable for the specific in vitro detection in a biological sample of S. enterica of group I, characterized in that it comprises the following oligonucleotides: SS2 (5'CCGGGCAGATGATACCC-3 'and SS28 ('-ATATGCTTTCCTGGTGC-3').   13. Protein IagA, characterized in that it is encoded by the nucleotide sequence iagA according to the claim 1.   14. Protein IagA according to claim 13, characterized in that it responds to the sequence of amino acids between the amino acids i and 553 of the sequence of FIG.   15. Protein IagB, characterized in that it is encoded by the sequence of nucleotides iagB according to the claim 1.   16. Protein IagB according to claim 15, characterized in that it responds to the sequence of amino acids between amino acids 554 and   713 of the sequence shown in FIG.   17. Process for the in vitro detection in a biological sample of Salmonella enterica nucleotide sequences, previously amplified, for example by PCR and denatured, characterized in that it comprises the steps of: denaturation of the S. enterica sequence amplified, - contacting the denatured amplified nucleotide sequences of S. enterica, with a capture probe and a revealing probe obtained from the oligonucleotides according to any one of   claims 2, 6 or 7 under conditions allowing   hybridizing said capture and revelation probes with the aforesaid amplified nucleotide sequence of S. enterica, the capture probe being attached to the surface of a well of a microtiter plate and the revealing probe being labeled and free in the hybridization buffer; incubating the reaction mixture for a time sufficient to allow the hybridization reaction; washing to remove unreacted oligonucleotides; - revelation of the revelation probes having   hybrid to the amplified nucleotide sequences.   18. The detection method as claimed in claim 17, characterized in that the capture probe is   tl'ol1i; gonleen + t i e T Ig At la s d ...- Ad1a, - - -. - - --- --7- ---- V .--., 41.   the oligonucleotide Iag3 and in that the detection is carried out according to the following steps: denaturation of a volume 10 1 of the amplified sequence are denatured by volume-to-volume addition of a solution of 200 mM NaOH, 40 mM EDTA, prehybridization of microplates, the well surfaces of which are coated with the capture probe, in a suitable hybridization buffer; - release of the microplate and filling of each of the wells with 200 μl of hybridization buffer containing the denatured amplified fragment and the revealing probe labeled with peroxidase at the concentration of 10 ng / 41, - incubation of the mixture for one hour at 37 ° C. with stirring, - washing of the reaction mixture with a washing solution 10 × (100 mM Tris, 3 M NaCl, 1% Tween 20, pH 7.4), - detection of the activity of the peroxidase linked to the probe by colorimetry in the presence of a colored substrate. 19. The detection method according to claim 18, characterized in that the activity of the peroxidase is detected by carrying out the following steps: depositing 20041 of a 40mM trisodium citrate solution, 0.03% H20 30%, 7.5 mg / ml of orthophenylenediamine (OPD) in each of the wells containing the reaction mixture, incubation of the microplate for 30 min in the dark and at 37 ° C, reaction blocking by adding 5041 / well of a 4N H2SO4 solution, determination of the optical density at a   wavelength of 492nm (reference 620nm).